(1) Field of the Invention
The invention relates to a laser scanning microscope (LSM), which scans a sample simultaneously with several spots and thus enables the image-recording time to be shortened. Such a microscope is described, for example, in U.S. Pat. No. 6,028,306, incorporated by reference herein. A device for generating multiple beams is described, for example, in U.S. Pat. No. 6,219,179, incorporated by reference herein.
(2) Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 1.98
With regard to the general construction of a confocal LSM, one can additionally refer to J. Pawley, “Handbook of biological confocal microscopy”, Springer (2006). In an LSM, fluorescing samples in particular are stimulated to emit light by at least one laser spot or a line, the spot or the line being scanned over the sample. The fluorescence, originating from the sample, is imaged on a confocal aperture, which effectively hides the light from non-focal planes of the sample. Light transmitted through an aperture is recorded with a photo detector and assembled by a computer by means of the location information from the laser positioning into a high-contrast fluorescing image of the respective focal plane. Three-dimensional image stacks can be produced by changing the focal plane.
The scanning process inherently takes a relatively long time to record the image. The approach, known from U.S. Pat. No. 6,028,306, of accelerating the rate of recording images confocally, is to scan the sample simultaneously with several excitation beams in small regions.
In this case, the confocal filtering is a critical parameter, especially with regard to the variability of the pinhole sizes.
Many proposed solutions simply specify fixed pinhole sizes. However, this is a disadvantage if the application, i.e., the customer, requires a large number of possible lenses, since the size of the fluorescence spots in the plane of the pinholes depends on the size of the lens pupil.
In addition, the distance between pinholes is then fixed, which places especially high adjusting and stability demands on the respective beam paths or does not allow any variability in the scanning area viewed.
Furthermore, if it is desired to switch over from a multifocal mode of operation and the conventional LSM operation, the maximum size of a pinhole is also always limited by the distance between the pinholes, which in this case is switchable.
In a confocal laser scanning microscope (LSM) with a multifocal excitation of the sample, complete confocality is to be established and adjusting tolerances of the individual beam paths or the relative positions of the confocal apertures to one another are not to result in pronounced brightness differences. This problem is solved by the features of the present invention.